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Hrpl Regulon of Bacterial Pathogen of Woody Host Pseudomonas Savastanoi Pv

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Hrpl Regulon of Bacterial Pathogen of Woody Host Pseudomonas Savastanoi Pv microorganisms Article HrpL Regulon of Bacterial Pathogen of Woody Host Pseudomonas savastanoi pv. savastanoi NCPPB 3335 Alba Moreno-Pérez 1,2, Cayo Ramos 1,2,* and Luis Rodríguez-Moreno 1,2,* 1 Área de Genética, Facultad de Ciencias, Campus Teatinos s/n, Universidad de Málaga, E-29010 Málaga, Spain; [email protected] 2 Departamento de Microbiología y Protección de Cultivos, Instituto de Hortofruticultura Subtropical y Mediterránea «La Mayora», Extensión Campus de Teatinos, Universidad de Málaga-Consejo Superior de Investigaciones Científicas (IHSM-UMA-CSIC), E-29010 Málaga, Spain * Correspondence: [email protected] (C.R.); [email protected] (L.R.-M.); Tel.: +34-952-132-146 (C.R.); +34-952-132-131 (L.R.-M.) Abstract: The Pseudomonas savastanoi species comprises a group of phytopathogenic bacteria that cause Symptoms of disease in woody hosts. This is mediated by the rapid activation of a pool of virulence factors that suppress host defences and hijack the host’s metabolism to the pathogen’s benefit. The hrpL gene encodes an essential transcriptional regulator of virulence functions, including the type III secretion system (T3SS), in pathogenic bacteria. Here, we analyzed the contribution of HrpL to the virulence of four pathovars (pv.) of P. savastanoi isolated from different woody hosts (oleander, ash, broom, and dipladenia) and characterized the HrpL regulon of P. savastanoi pv. savastanoi NCPPB 3335 using two approaches: whole transcriptome Sequencing (RNA-seq) and the bioinformatic prediction of candidate genes containing an hrp-box. Pathogenicity tests carried out for the P. savastanoi pvs. showed that HrpL was essential for symptom development in both Citation: Moreno-Pérez, A.; Ramos, non-host and host plants. The RNA-seq analysis of the HrpL regulon in P. savastanoi revealed a C.; Rodríguez-Moreno, L. HrpL Regulon of Bacterial Pathogen of total of 53 deregulated genes, 49 of which were downregulated in the DhrpL mutant. Bioinformatic Woody Host Pseudomonas savastanoi prediction resulted in the identification of 50 putative genes containing an hrp-box, 16 of which pv. savastanoi NCPPB 3335. were Shared with genes previously identified by RNA-seq. Although most of the genes regulated Microorganisms 2021, 9, 1447. https:// by HrpL belonged to the T3SS, we also identified some genes regulated by HrpL that could encode doi.org/10.3390/microorganisms9071447 potential virulence factors in P. savastanoi. Academic Editors: Rafael Rivilla and Keywords: P. savastanoi pv. savastanoi (Psv); type III secretion system (T3SS); HrpL regulon; RNA-seq Jacob G. Malone analysis; hrp-box prediction; virulence factors Received: 9 June 2021 Accepted: 29 June 2021 Published: 5 July 2021 1. Introduction The type III secretion system (T3SS) is considered one of the most relevant virulence Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in mechanisms in animal and plant pathogenic bacteria. The T3SS is a complex membrane- published maps and institutional affil- embedded nanomachine through which Gram-negative bacteria translocate a set of pro- iations. teins, known as type III effectors (T3Es), into the cytoplasm of host cells [1–7]. Structurally, the injectosome is made up of more than 20 proteins and is the most complex secre- tion system in bacteria [8]. From a functional point of view, the translocation of T3Es contributes to perturbing host cellular functions to facilitate bacterial survival and host colonization [6,9–13]. The T3SS is essential for Pseudomonas syringae pathogens to thrive Copyright: © 2021 by the authors. in plant tissues. Although the evolution of the T3SS remains controversial, phylogenetic Licensee MDPI, Basel, Switzerland. This article is an open access article analysis through amino acid sequence comparison suggests that the T3SS first emerged in distributed under the terms and plant pathogens as an evolutionary adaptation of the flagellar export apparatus [4]. conditions of the Creative Commons The P. syringae species complex is considered one of the most relevant phytopathogenic Attribution (CC BY) license (https:// bacteria worldwide, due to its capacity to infect the phyllosphere and cause disease in a creativecommons.org/licenses/by/ diverse range of cultivated, ornamental, and wild plants [14,15]. The complex comprises 4.0/). 13 phylogroups (PGs) encompassing 15 Pseudomonas species [16,17] that can be divided Microorganisms 2021, 9, 1447. https://doi.org/10.3390/microorganisms9071447 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, 1447 2 of 21 into about 65 pathovars (pv.) defined by their host ranges [18]. Except for some naturally occurring non-pathogenic P. syringae strains that lack the canonical T3SS [19–21], most of the Strains included in the P. syringae complex require a functional T3SS for pathogenesis in susceptible plants [4,22,23]. The T3SS in P. syringae is encoded and regulated by the products of the hypersensi- tive response and pathogenicity (hrp) and hypersensitive response and conserved (hrc) gene clusters, which are included in a tripartite pathogenicity island together with other genes that encode accessory and conserved T3Es [24,25]. After translocation into the host cytoplasm, T3Es subvert host cellular functions, facilitating bacterial survival and host colonization [6,9–13]. The recognition of T3Es or their activity by the plant immune Sys- tem, through resistance proteins or other mechanisms, induces the host’s hypersensitive response (HR), a localized plant cell death response that limits bacterial growth [26]. For this reason, the T3SS and its T3E repertoire have been recognized as the main determinants of host specificity in P. syringae [27–29]. The transcriptional regulation of T3SS’ structural components and their associated T3E repertoire in P. syringae is dependent on the HrpL regulator, which encodes an alter- nate Sigma (σ) factor that recognizes a conserved promoter sequence (GGAACC-N15/16- CCACNNA), known as the hrp-box [30]. HrpL’s expression depends on the σ54 factor RpoN and two transcriptional activators, HrpR and HrpS, which work as heterodimers and cooperate with σ54 to promote the expression of hrpL [31,32]. Recent studies have clearly shown that the Signaling pathways and molecular mechanisms involved in T3SS regulation in P. syringae are a complex, intricate network [33] involving dozens of regulatory proteins [8,34], second messenger molecules such as c-di-GMP [35], and variations in the physicochemical conditions during host colonization [36]. Pseudomonas savastanoi belongs to the PG3 group of the P. syringae complex; the unique PG includes knot-producing bacteria in woody hosts [16,37,38]. P. savastanoi comprises five pathovars that cause diseases in woody plants: pv. savastanoi (Psv, isolated from olive), pv. nerii (Psn, isolated from oleander), pv. fraxini (Psf, isolated from ash), pv. retacarpa (Psr, isolated from broom), and pv. mandevillae (Psm, isolated from dipladenia) [37,39]. In the Psv and Psn strains, the functionality of the T3SS has been shown to be essential for knot formation in the respective hosts and the induction of a characteristic HR in resistant hosts [37,40–43]. Recent comparative genomics analysis of strains belonging to these five pathovars identified the codification of highly conserved canonical T3SSs in strains of Psf, Psm, and Psr. However, their functionality and roles in pathogenesis in these three pathovars have not yet been established [39,44,45]. Furthermore, and as previously reported for Psv NCPPB 3335 [46], an additional T3SS resembling that found in Rhizobiaceae was also found in the other pathovars [39,44]. The relevance of T3SS regulation by HrpL in P. savastanoi is evidenced by the inability to induce knot formation in olive plants and to induce HR in tobacco plants by using a DhrpL mutant of the model Psv strain NCPPB 3335 [47]. Furthermore, pathovar-specific regulation of the T3SS and its T3E genes has been identified in Psv, Psn, and Psf, suggesting a possible role in host range, depending on the physiological conditions found in the apoplast, or the extracellular space of the host plant tissues [45]. The global regulation of transcription by HrpL in the P. syringae complex has been approached using microarrays or RNA-seq strategies. However, the HrpL regulon has only been defined in P. syringae strains isolated from herbaceous hosts [48,49], and no data are available for strains isolated from woody hosts. Here, we constructed HrpL mutants of model Psn, Psm, Psf, and Psr strains to study the role of this regulator in the pathogenicity of P. savastanoi strains isolated from other woody hosts. Then, we defined the HrpL regulon of Psv NCPPB 3335, the only P. savastanoi strain whose chromosome [44,46] and plasmids [50] have been fully sequenced. For this purpose, we used two approaches: (i) the comparative transcriptomic analysis (RNA-seq) of wild-type Psv NCPPB 3335 and its DhrpL mutant, and (ii) the bioinformatic prediction of hrp-box promoters in the genome of this strain. A comparison of the results obtained from these analyses with those Microorganisms 2021, 9, 1447 3 of 21 previously reported for P. syringae strains isolated from herbaceous hosts allowed us to unravel novel HrpL-dependent genes that may play a role in the virulence of P. savastanoi and the interactions of bacterial pathogens with woody hosts. 2. Materials and Methods 2.1. Bacterial Strain, Plasmids, and Growth Conditions The bacterial strains and plasmids used in this study are described in Tables S1 and S2, respectively. All the P. savastanoi strains were grown at 28 ◦C in lysogeny broth (LB) medium [51] without glucose and containing 0.5% NaCl, in King’s B (KB) medium [52] or in Super Optimal Broth (SOB) medium [53]. Escherichia coli strains were grown in LB medium at 37 ◦C. When required, the medium was supplemented with the following: for P. savastanoi: ampicillin (Ap) (400 µg/mL), kanamycin (Km) (7 µg/mL), nitrofurantoin (Nf) (25 µg/mL), and cycloheximide (Ch) (100 µg/mL); for E. coli: Ap (100 µg/mL) and km (50 µg/mL).
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